HAL Id: pasteur-00734481
https://hal-riip.archives-ouvertes.fr/pasteur-00734481
Submitted on 22 Sep 2012
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Emergence of quinolone resistance among extended-spectrum beta-lactamase-producing Enterobacteriaceae in the Central African Republic:
genetic characterization.
Thierry Frank, Jean R Mbecko, Pembe Misatou, Didier Monchy
To cite this version:
Thierry Frank, Jean R Mbecko, Pembe Misatou, Didier Monchy. Emergence of quinolone resis- tance among extended-spectrum beta-lactamase-producing Enterobacteriaceae in the Central African Republic: genetic characterization.. BMC Research Notes, BioMed Central, 2011, 4, pp.309.
�10.1186/1756-0500-4-309�. �pasteur-00734481�
S H O R T R E P O R T Open Access
Emergence of quinolone resistance among extended-spectrum beta-lactamase-producing
Enterobacteriaceae in the Central African Republic:
genetic characterization
Thierry Frank
*, Jean R Mbecko, Pembe Misatou and Didier Monchy
Abstract
Background: Cross-resistance to quinolones and beta-lactams is frequent in Enterobacteriaceae, due to the wide use of these antibiotics clinically and in the food industry. Prescription of one of these categories of antibiotic may
consequently select for bacteria resistant to both categories. Genetic mechanisms of resistance may be secondary to a chromosomal mutation located in quinolone resistance determining region of DNA gyrase or topoisomerase IV or to a plasmid acquisition. The insertion sequence ISCR1 is often associated with qnr and may favour its dissemination in Gram-negative bacteria. The aim of this study was to determine the genetic mechanism of quinolone resistance among extended-spectrum beta-lactamase-producing Enterobacteriaceae strains in the Central African Republic.
Findings: Among seventeen ESBL-producing Enterobacteriaceae isolated from urine, pus or stool between January 2003 and October 2005 in the Central African Republic, nine were resistant to ciprofloxacin (seven from community patients and two from hospitalized patients). The ESBL were previously characterized as CTX-M-15 and SHV-12.
Susceptibility to nalidixic acid, norfloxacin and ciprofloxacin, and the minimal inhibitory concentrations of these drugs were determined by disc diffusion and agar dilution methods, respectively. The presence of plasmid-borne ISCR1-qnrA region was determined by PCR and amplicons, if any, were sent for sequencing. Quinolone resistance determining region of DNA gyrase gyrA gene was amplified by PCR and then sequenced for mutation
characterization. We found that all CTX-M-producing strains were resistant to the tested quinolones. All the isolates had the same nucleotide mutation at codon 83 of gyrA. Two Escherichia coli strains with the highest MICs were shown to harbour an ISCR1-qnrA1 sequence. This genetic association might favour dissemination of resistance to quinolone and perhaps other antibiotics among Enterobacteriaceae.
Conclusions: This study shows that at least two mechanisms might explain the emerging resistance of Enterobacteriaceae to quinolones in the CAR. Beside the classical topoisomerase mutation, the cause may be acquisition of a plasmid-borne qnrA1. Clinicians and bacteriologists should be made aware of possible dissemination of ISCR1-qnrA1 among Enterobacteriacae.
Keywords: Quinolones, Betalactams, Antibiotic resistance, Enterobacteriaceae
Background
Quinolones and beta-lactams are widely used antibiotics for the treatment of bacterial infections and are also extensively used in the food-processing industry. Their wide use has triggered increased bacterial resistance
worldwide [1]. Quinolone resistance and extended-spec- trum beta-lactamase (ESBL) production are often asso- ciated in Enterobacteriaceae [1]. The prescription of quinolones may consequently select bacteria that are not only quinolone-resistant but also beta-lactam-resis- tant and vice versa. The biological mechanisms of resis- tance to quinolones include impermeability, active efflux, target modification and antibiotic neutralization.
* Correspondence: simplicefrank@gmail.com
Service de Bactériologie, Institut Pasteur de Bangui, Bangui, Central African Republic
Franket al.BMC Research Notes2011,4:309 http://www.biomedcentral.com/1756-0500/4/309
© 2011 Frank et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genetic determination of quinolone resistance is gener- ally linked to chromosomal mutations affecting the qui- nolone resistance-determining regions (QRDR) of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) [1]. Mutations in gyrA, the gene encoding for the A subunit of DNA gyrase are the most common mechanism involved in quinolone resistance among Gram negative bacteria [2].
In 1994, the plasmid-borne quinolone resistance genes qnr were identified [1]. The insertion sequence ISCR1 has often been characterized in the genetic environment of qnr and may be involved in dissemination of the gene in Gram-negative bacteria [3].
Quinolone resistance was first identified in the CAR in 2003, among clinical ESBL-producing Enterobacteria- ceae. The aim of this study was to determine the genetic mechanism of quinolone resistance of these strains iso- lated in the Central African Republic (CAR).
Methods
Of the seventeen ESBL-producing Enterobacteriaceae isolated from urine, pus or stool samples between Janu- ary 2003 and October 2005 at the Institut Pasteur de Bangui and previously characterized as CTX-M-15 and SHV-12, nine were resistant to ciprofloxacin (eight Escherichia coli and one Klebsiella pneumoniae) [4].
Seven E.coli strains were isolated from community patients and the two others were isolated from hospita- lized patients.
Antibiotic susceptibility was determined by the diffu- sion disc method (Comité de l’Antibiogramme de la Société Française de Microbiologie). The minimal inhi- bitory concentrations (MICs) of nalidixic acid, norfloxa- cin and ciprofloxacin were measured by the agar dilution method.
PCR was carried out on genomic DNA extracted with the commercial Qiagen DNA Mini Kit (Qiagen, Courta- boeuf, France) at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 55 °C for 40 s and extension at 72 °C for 60 s, fol- lowed by 72 °C for 7 min.
The ciprofloxacin-resistant isolates were screened for the qnrA gene by PCR with specific primers targeting ISCR1, 5’-TGAAACAGAAAACAGCCAAGG-3’ (ISC R1qnr upper), and qnrA, 5’-GCAGCACTATTACTC- CAAGG-3’ (ISCR1qnr lower). The primers targeting ISCR1-qnrA1 were designed with Oligo-4 software. The sequences encoding QRDR of DNA gyrase were ampli- fied with gyrA6 5 ’ -CGA CCT TGC GAG AGA AAT-3 ’ and gyrA631R 5 ’ -GTT CCA TCA GCC CTT CAA-3 ’ [5].
All amplicons were sequenced at MWG Biotech France (http://www.mwg-biotech.com) and analysed with program ChromasPro 2.0 and blastN software
(http://gb-admin@ncbi.nlm.nih.gov) (GenBank accession numbers for the sequenced ISCR1-qnrA1 region are JF728153 and JF728154).
All patients have given their agreement to participate in this study prior the samples collection. This study was reviewed and approved by the scientific committee board of University of Bangui in charge of the validation of scientific protocol studies in the CAR.
Results and discussion
Susceptibility testing showed that all CTX-M-producing strains were resistant to nalidixic acid, norfloxacin and ciprofloxacin. The MIC of nalidixic acid and norfloxacin was 64 μg/ml and that of ciprofloxacin was 32 μg/ml for six E. coli and the K. pneumoniae strains. For two E. coli isolates, the MICs were higher, and PCR targeting ISCR1-qnrA amplified a 1050-bp fragment from these two isolates which was shown by DNA sequence analy- sis to be ISCR1-qnrA1. We could not determine the MICs values of the two qnrA-positive E. coli by the agar dilution method because they exceeded the highest con- centrations of antibiotics tested in this study (32 μ g/ml for ciprofloxacin and 64 μ g/ml for the nalidixic acid).
Other publications have shown that qnrA increased the MICs of quinolones by 10-fold in Enterobacteriaceae [6]. The combination of an insertion sequence ISCR1 with qnrA1 could ensure dissemination of quinolone resistance to both ESBL-producing and ESBL-negative Enterobacteriaceae. The ISCR1 element is also capable of mobilizing qnrA1 in multiple drug resistance regions, generally located on plasmids that mediate resistance to multiple antibiotics.
Sequence analysis of the gene portion encoding the QRDR of GyrA showed the same amino acid change in GyrA (Ser83-Leu) in all our isolates. Nucleotide muta- tions at codon 83 are the commonest GyrA substitu- tions found in quinolone-resistant E. coli and the serine residue is most commonly substituted by a leucine [7].
Conclusion
We have shown that the emerging resistance of Entero- bacteriaceae to quinolones in the CAR may be due to a topoisomerase mutation or a plasmid-borne qnrA1 acquisition. It would be interesting to sequence the DNA gyrase gyrB and topoisomerase IV parC and parE genes, to screen for the other qnr genes (B and S) and to characterize the environment of ISCR1-qnrA1, which has already been found in several sul1-type complex integrons. The possible dissemination of ISCR1-qnrA1 among Enterobacteriacae requires vigilance by bacteriol- ogy laboratories in the CAR. It will also be important to inform clinicians of the potential risk associated with prescribing quinolones and beta-lactams.
Franket al.BMC Research Notes2011,4:309 http://www.biomedcentral.com/1756-0500/4/309
Page 2 of 3
Acknowledgements and funding
We thank Vianney Tricou, Mirdad Kazanji and Elisabeth Heseltine for their critical reading of the manuscript. This work was financed by the French government and the European Union.
Authors’contributions
TF: carried out the molecular genetic studies, validated the results of antibiotic susceptibility.testing, participated in nucleotide sequence analysis and drafted the manuscript.
JRM: participated in bacteriological isolation ofEnterobacteriaceaestrains. PM:
participated in bacteriological isolation ofEnterobacteriaceaestrains. DM:
helped to draft the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 18 April 2011 Accepted: 25 August 2011 Published: 25 August 2011
References
1. Ambrozic Avgustin J, Keber R, Zerjavic K, Orazem T, Grabnar M:Emergence of the quinolone resistance-mediating geneaac(6’)-Ib-crin extended- spectrum-beta-lactamase-producing Klebsiella isolates collected in Slovenia between 2000 and 2005.Antimicrob Agents Chemother2007, 51:4171-4173.
2. Gruger T, Nitiss JL, Maxwell A, Zechiedrich EL, Heisig P, Seeber S, Pommier Y, Strumberg D:A mutation in Escherichia coli DNA gyrase conferring quinolone resistance results in sensitivity to drugs targeting eukaryotic topoisomerase II.Antimicrob Agents Chemother2004, 48:4495-4504.
3. Jacoby GA:Mechanisms of resistance to quinolones.Clin Infect Dis2005, 41(Suppl 2):S120-126.
4. Frank T, Arlet G, Gautier V, Talarmin A, Bercion R:Extended-spectrum beta- lactamase-producing Enterobacteriaceae, Central African Republic.Emerg Infect Dis2006,12:863-865.
5. Lavilla S, Gonzalez-Lopez JJ, Sabate M, Garcia-Fernandez A, Larrosa MN, Bartolome RM, Carattoli A, Prats G:Prevalence ofqnrgenes among extended-spectrum beta-lactamase-producing enterobacterial isolates in Barcelona, Spain.J Antimicrob Chemother2008,61(2):291-295.
6. Lascols C, Robert J, Cattoir V, Bebear C, Cavallo JD, Podglajen I, Ploy MC, Bonnet R, Soussy CJ, Cambau E:Type II topoisomerase mutations in clinical isolates of Enterobacter cloacae and other enterobacterial species harbouring the qnrA gene.Int J Antimicrob Agents2007, 29:402-409.
7. Hopkins KL, Davies RH, Threlfall EJ:Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments.Int J Antimicrob Agents2005,25:358-373.
doi:10.1186/1756-0500-4-309
Cite this article as:Franket al.:Emergence of quinolone resistance among extended-spectrum beta-lactamase-producing
Enterobacteriaceaein the Central African Republic: genetic characterization.BMC Research Notes20114:309.
Submit your next manuscript to BioMed Central and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at www.biomedcentral.com/submit Franket al.BMC Research Notes2011,4:309
http://www.biomedcentral.com/1756-0500/4/309
Page 3 of 3
